Four oyster mushroom species are cultivated in North Bengal for its good taste, texture and therapeutic values. Morphological and anatomical characteristics of four Pleurotus species (P. djamor, P. sajorcaju, P. ostreatus and P floridanus) have been elucidated. Malt extract agar showed highest mycelial growth rate followed by the potato dextrose agar, while water agar was lowest. All four Pleurotus species were identified using 18S rDNA sequencing as P. djamor (KT768094), P. sajor-caju (KT818506), P. ostreatus (KT768095) and P floridanus (KT826605) and subsequently all the sequence have been deposited in the NCBI Genbank. 18S r DNA sequence alignments of P. djamor, P. sajor-caju, P. ostreatus and P floridanus with other ex-type isolates have been presented and the conserved regions of the gene have been demonstrated in different colours. Phylogenetic placement of all four Pleurotus species with other 41 ex-type strains obtained from NCBI GenBank was compared using UPGMA method in MEGA11 software and result has been presented. Antioxidant activity such as DPPH scavenging activity, ferric reducing antioxidant power activity as well as flavonoid activity of four Pleurotus species were also evaluated. P. djamor and P. ostreatus showed higher DPPH scavenging activity in comparison to other two species. Highest amount of reducing power activity was observed in P. djamor and lowest in P. ostreatus and P. sajor-caju. P. djamor and P. floridanus also showed higher flavonoid content in comparison to other two species.