Hand hygiene is a vital principle and exercise in the prevention, control and reduction of healthcare acquired infections. A practise of hand sanitizing is very important to eliminate the microbial contamination especially work in laboratories, hospital and even at home. Proper use of hand sanitizer reduces the transmission of infection also. Herbal hand sanitizer was prepared by using herbs like Neem (0.3%), Turmeric (0.2%), Ginger (0.2%) Lemon (0.01%), and Mint (0.01%). The surface swabs were collected from various places of STET Campus. From the sample predominant organisms were isolated such as E.coli, P.aeroginosa, S.aureus and fungal species such as Candida albicans. The isolated organisms tested with herbal hand sanitizer at different concentrations such as 25%, 50% and 100% by using agar well diffusion method. Among these 100% of herbal hand sanitizer showed the highest zone diameter was observed as (65±5.3) against E.coli. It is concluded that herbal hand sanitizer has a significant antimicrobial effect on isolated microorganisms. The further study is to be planned to formulate the herbal hand sanitizer in gel form also.