The alkaline Lonar Lake is a unique ecosystem formed by meteor impact, situated in the Buldhana District, Maharashtra, India. The Lonar Lake harbours diversified microbial flora which can detoxify and degrade most harmful pollutant such as methanol. The Methylotroph which degrade harmful methanol can produced bioactive substances having antimicrobial potentials. The two methylotrophic Pseudomonas species were isolated and characterized by cultural, morphological, biochemical tests and by 16S rRNA gene sequencing and identified as P. aerogenosa (DHT2) and P. hibiscola (DHT 11). All these selected Pseudomonas species exhibited antimicrobial activities against pathogenic bacteria and data showed that and P. aerogenosa were stronger antimicrobial than to P. hibiscola. The Present study provides primary evidence that isolated Pseudomonas species were promising sources for production of antimicrobial bioactive substances and represent a new and rich source of secondary metabolites that need to be explored in medical microbiology.