Trichoderma spp. produce diverse secondary metabolites with biological activity. This study explored the antimicrobial, antibiofilm, antioxidant, and cytotoxic properties of metabolites from two native Trichoderma strains, 10BR1 and UEPA AR12, isolated from rhizospheric soils. Organic extracts from both strains demonstrated broad-spectrum antimicrobial activity, inhibiting Gram-positive and Gram-negative bacteria, as well as various Candida species, with notable efficacy against Staphylococcus aureus (MICs: 15.6–31.25 µg/mL). The extracts also showed antibiofilm activity, with UEPA AR12 exhibiting the highest inhibition against Escherichia coli (81.8%), Enterococcus faecalis (92.8%), Candida albicans (87.9%), and Candida parapsilosis (89.3%). Antioxidant activity, assessed via DPPH assay, revealed a dose-dependent radical scavenging effect (12.88% to 39.67% at 7.8–1000 µg/mL). Cytotoxicity assays indicated that UEPA AR12 extracts were more cytotoxic (IC₅₀: 202.5–234.3 µg/mL) than 10BR1 (IC₅₀: 368.7–602.1 µg/mL) in non-tumor cells, with similar trends in tumor cells (Huh7). HPLC/MS analysis identified 21 metabolites in the extracts. Genomic analyses, supported by rpb2 gene and phylogenetic clustering, confirmed that both strains were T. afroharzianum. FUNGISMASH revealed multiple biosynthetic gene clusters, predominantly Type I polyketide synthase (T1PKS). Additionally, targeted genomic analyses did not detect mycotoxin-related genes. These findings highlight the antimicrobial, antibiofilm, and antioxidant potentials of these strains, positioning them as sources of bioactive metabolites for pharmaceutical applications.