The large-scale identification and quantitation of proteins via nanoliquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate (SDS) protein extraction + 1D gel LC; phenol protein extraction + 1D gel LC] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85-15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (≥1 peptide) or 54.7% (≥2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.