Microbial asymmetric reduction of ketone is an efficient tool for the synthesis of chiral alcohols. Many reports are available for the bio-reduction of acetophenone and its derivatives, since it is widely used model substrate. This research focus on exploring the soil fungal isolates for their ability towards the reduction of acetophenone and its derivatives to their corresponding chiral alcohols using growing cells instead of resting cells. Bio-reduction of acetophenone, 4-fluoro acetophenone, and 4-chloro acetophenone were carried out using different fungal cultures isolated from soil. All the isolates exhibited a good reduction capability when grown in an optimal condition. Among the screened fungal cultures, Penicillium sp. and Aspergillus sp. showed significant bioconversion with varying enantio-selectivity. However, the Penicillium sp. has showed maximum ability of bio-reduction. The best performing isolate was characterized using internal transcribed spacer (ITS) region and found to be Penicillium rubens VIT SS1, which showed higher conversion and selectivity more than 90% towards acetophenone and its derivatives. The reaction conditions such as pH, temperature and media were evaluated for the bio-reduction of acetophenone using Penicillium rubens VIT SS1. The substrate loading was increased from 0.5g/L to 6g/L at shake flask level using the optimized condition pH 5±0.5 and temperature 25±2°C. This study revealed huge potential of fungal cultures for the synthesis of many aromatic chiral alcohols in a simpler, novel and cost effective manner. © 2019 IJSTR.