The Micropropagation, Callogenesis and Antimicrobial activity of in vivo and in vitro grown plants of Amaryllis belladonna was studied. The shoot regeneration was best at BAP-3 mg/l. Minimum time for multiple shoot formation was shown by BAP-3 while best multiple shooting results were shown by BAP-5 mg/l. NAA along with BAP has evident effect on root number as well as root length. In vitro grown plants were then established in growth chamber and after that in green house to acclimatize. The antimicrobial activity of in vivo and in vitro grown plants was compared, bulb extract showed maximum response against bacterial as well as the fungal strains as compared to leaf extracts. In vitro bulb extracts were more effective against bacterial strains as compared to in vivo bulb extract and the maximum zone of inhibition was shown against Bacillus licheniformis. In vivo bulb extract showed better effect as compared to in vitro bulb extract and the maximum zone of inhibition was shown against Aspergillus niger.