Biotransformations catalysed by microbes are promising approach for producing a vast library of structurally diverse chemical molecules with applications in the pharmaceutical industry. The biotransformation of 11α-acetoxyprogesterone (1) by Phyllosticta sp. 16L1 has not been previously reported. In this study, the biotransformation of 11α-acetoxyprogesterone (1) was performed for the first time using the Phyllosticta sp. 16L1 strain. After an 8-day fermentation period, a new biotransformation metabolite, named as 11α-acetoxy-16α-hydroxyprogesterone (16α-hydroxy-3,20-dioxopregn-4-en-11α-yl acetate) (2) was isolated from the culture broth, along with its known isomer, 11α-acetoxy-15α-hydroxyprogesterone (3). The structure determination of the biotransformed products relied on comprehensive spectroscopic data, encompassing 1D and 2D-NMR, as well as LCMS analyses. The cytotoxic activity of the two biotransformed metabolites was assessed against selective human cancer cell lines, including hepatocellular carcinoma (HepG2), triple-negative breast cancer (MDA-MB-231), colorectal adenocarcinoma (Caco-2), and lung adenocarcinoma (A549). The results demonstrated that both metabolites 2 and 3 exhibited cytotoxic effects on the evaluated cell lines. Metabolite 2 showed stronger cytotoxic potential, with IC