Background/Objectives: Antimicrobial resistance and biofilm-associated infections have intensified the search for new bioactive molecules from natural sources. This study evaluated the antibacterial activity, effects on biofilm biomass, preliminary cytotoxicity, and chemical profiles of ethyl acetate extracts obtained from Amazonian endophytic fungi against Staphylococcus aureus CCCD-S009. Methods: Ten fungal extracts were evaluated using broth microdilution and crystal violet-based biofilm assays. Additional experiments assessed biofilm biomass during formation and the reduction in preformed biofilm biomass at the minimum inhibitory concentration (MIC) and half the MIC (0.5× MIC). Complementary experiments with Talaromyces pinophilus CCM-UEA-F0408 included testing across multiple concentrations and qualitative microscopy. Cytotoxicity was evaluated in MRC-5 human fibroblasts, and chemical characterization included thin-layer chromatography, total phenolic content determination, and HPLC-PDA analysis of selected phenolic acids. Results: Nine extracts showed antibacterial activity, with Cophinforma mamane CCM-UEA-F0869 and Penicillium jenningsiae CCM-UEA-F0773 exhibiting the lowest MICs (312.5 µg/mL). Nine extracts reduced crystal violet-stained biofilm biomass during formation by more than 50%. At 0.5× MIC, reductions in biofilm biomass ranged from 73.40% to 86.94%. Removal of preformed biofilm biomass was less consistent and varied with extract concentration and exposure time. At 50 µg/mL, seven extracts maintained MRC-5 cell viability above 90%, whereas T. pinophilus CCM-UEA-F0695 and P. jenningsiae CCM-UEA-F0773 markedly reduced cell viability. Preliminary chemical analyses revealed differences among extracts, and targeted HPLC-PDA profiling indicated the presence of caffeic, 4-hydroxybenzoic, gallic, and vanillic acids in selected fungal extracts. Conclusions: Amazonian endophytic fungal extracts exhibited distinct antibacterial, biofilm-related, and cytotoxicity profiles. The reduction in biofilm biomass observed at sub-MICs supports further investigation of these extracts as potential sources of bioactive compounds. However, biofilm-specific effects cannot be distinguished from growth inhibition using the present assays alone.