The deep subseafloor biosphere represents a frontier for the discovery of new microbial life and for investigations of the extent, versatility, and perseverance of life on earth. However, there are many challenges in studying this community of microorganisms, and the past 20 years of study have only begun to shed light on this vast and complex ecosystem. With each chapter herein I have taken on some of those challenges and have made progress in overcoming them, while also contributing to the knowledge of an environment that—despite its potential significance—remains relatively unexplored. In particular I have focused on the application of molecular methods to the study of the subseafloor biosphere, which is complicated by difficulties such as low biomass, extracellular and fossil DNA, potential for drilling-induced contamination, and method biases. In chapter 2, I examined the potential sources of molecular signals suggestive of phototrophic organisms in the subseafloor via cultivation, DNA sequencing, and PCR-based inquiries. Although I found that most likely the molecular signals of phototrophic organisms found in the deep biosphere do not represent viable cells, factors such as the uncertainty of DNA survival time and the paucity of information on many subseafloor taxonomic lineages made it difficult to furnish an explanation for these molecular signals. Additionally, while I was unable to succeed in demonstrating phototrophy in cultivations, my results suggested that I was able to stimulate other microbial growth, and that in most cases the organisms that became dominant in the cultivations had been only a minor proportion of the original uncultivated sediment. This is a good demonstration of the potential importance of even the “minor” components of a microbial community under changing environmental conditions. In chapter 3, I took on the challenges of carrying out molecular work on very low biomass sediment samples by developing and testing a novel method of whole genome amplification that overcame some of the limitations of previous methods for subseafloor samples. While the method solved some problems specific to low biomass samples and seemed a viable alternative to previous methods of whole genome amplification for these samples, my work reaffirmed previous studies in showing that there are still dangers in interpreting community data based on DNA that has been subjected to whole genome amplification with any method. Further, I identified problems with comparing data from different sequencing technologies and with different data analysis and classification methods. In chapter 4, I carried out a follow-up to the study of whole genome amplification utilizing samples from 2 previously uncharacterized subseafloor locations in the Eastern Equatorial Pacific. While the limitations of utilizing amplified DNA were again reinforced, my results showed that at some levels community analysis on amplified DNA was relatively accurate. Here I was able to show a robust taxonomic distinction between these 2 new sites from the pelagic abyss and metagenomes from 3 previously available coastal margin subseafloor locations, while also demonstrating the potential validity of predicting microbial community composition in a subseafloor location based on results from a nearby and very similar subseafloor location. In the final chapter I took on the problem of drilling-induced contamination by carrying out the first study of its kind to extensively characterize the microbial community from both the sediments and the corresponding drilling fluid used during sample acquisition, on a range of sample depths including several samples taken with the more aggressive extended core barrel coring (XCB) method. I found the drilling fluid to have a very minor influence in the molecular analysis of all samples except for one, which lends confidence to the study of deep cores while at the same time reinforcing the importance of making drilling fluid controls a standard part of every molecular study of subseafloor sediment samples. Additionally, I characterized the previously unexplored microbial communities of the Costa Rica Margin subseafloor and discussed some potential linkages between subseafloor microbial taxa and pore-water geochemistry variables. Considering the current state of knowledge in this environment due to its challenging nature, the work herein contributes greatly to our understanding of microbial biogeography and relationships with environmental conditions, as well as to the many complexities in performing and interpreting molecular analyses in the subseafloor. It also provides a wealth of new 16S rRNA and metagenomic datasets that can continue to be used for further investigations.