The biopolymer melanin is reported for many biological processes to secure biological entities over unfavorable environmental factors. The present study aimed to isolate soil fungi and screen for melanin production. The potent fungus was identified as Penicillium citrinum NP4 based on morphological and molecular characterization with accession number OP070954. Using standardized tyrosine broth conditions melanin was produced by NP4 and extracted by acidification. Extracted melanin exhibited maximum UV-visible absorption at 223 nm; FTIR peaks validate the occurrence of CO, CN, CH, and CC functional groups present in the indole/pyrrole structure. TLC analysis exhibited a prominent single band with a Retardation factor (Rf) of 0.68, resonance peaks at 6.621, 7.061, and 7.185 ppm exhibited aromatic hydrogen in the indole/pyrole system in H NMR. The EDX peaks confirm the presence of carbon, oxygen, sulfur, and nitrogen elements which are the key factors in melanin structure, and TGA reports the thermal stability of the melanin. An in silico molecular docking approach on lung cancer causing proteins EGFR (3g5z), KRAS (6vc8), and TP53 (8 dc4) were conducted to determine the active binding sites of the melanin, and proteins exhibited binding affinity of -8.0 for 3g5z, -9.8 for 6vc8, and - 10.1 kcal/mol for TP53 protein with melanin. Anticancer activity of the melanin showed significant inhibition of A549 cells in dose-dependent mode with significant IC of 65.49 μg/mL; apoptotic examination reveals 46.14 % apoptosis for melanin and 46.36 % apoptosis for standard drug (cisplatin). Melanin exhibited good photoprotection capacity at 1 μg/mL. In conclusion, the extracted melanin exhibited significant results on many biological applications and it can be used in the pharmaceutical field to avoid chemical-based drugs.