This study gold (Au) and gold–platinum (Au–Pt) colloids were synthesized by borohydride reduction method in the presence of poly(N-vinyl-2-pyrrolidone) as a stabilizing agent. Besides, pristine polyaniline (PANI), PANI-Au nanocomposites, and PANI-Au–Pt nanocomposites were prepared. The synthesized nano-composites characterized by UV–Vis, FT-IR, XRD, and HR-TEM with energy dispersive X-ray techniques. Au and Au–Pt NPs were spherical in shape with particle sizes at 8 and 3 nm, respectively. PANI-Au and PANI-Au–Pt nanocomposites were also spherical in shape with particle sizes at 3 and 2 nm, respectively. Pristine PANI, PANI-Au, and PANI-Au–Pt nanocomposites were subjected to antibacterial activity against gram-positive (Bacillus subtilis and Staphylococcus aureus) and gram-negative (Escherichia coli and Vibrio cholerae) bacterial pathogens. PANI-Au–Pt nanocomposite showed significant antibacterial activity (33 ± 1.10 mm dia) against B. subtilis. Besides, the MIC of pristine PANI, PANI-Au, and PANI-Au–Pt nanocomposites were found to be 75, 50 and 25 µg/mL, respectively. Besides, the in vitro anticancer investigations against HepG2 liver cancer cells revealed the highest cytotoxicity for PANI-Au–Pt nanocomposite (21.25 µg/mL) followed by PANI-Au nanocomposite (32 µg/mL) and pristine PANI (49 µg/mL). Overall, the present study showed considerable antibacterial and anticancer activity and suggested their potential use in pharmaceuticals after completing successful clinical trials for safety and affordability.