Present study was conducted to evaluate the impact of ozonation on the quality improvement of Secondary Treated wastewater at Varanasi, in India. Physico-chemical parameters such as BOD, COD, TOC, UV 254 , color and microbiological parameters such as E. coli, fecal coli. & total coliform disinfection parameters were used to evaluate the efficiency of ozone to upgrade secondary effluent quality. Experimental results revealed that 10 mgL -1 O 3 for 5 minutes exposure duration was found most suitable dose and contact time for the significant reduction of physicochemical parameters of secondary treated wastewater such as BOD, COD, TOC, UV 254 and color i.e. 30%, 14.3% 45% & 60% respectively as well as it enhanced the biodegradability more than 50% in the treated water. Our data also revealed that this applied dose and exposure time is responsible for the highest degradation of microbiological parameters i.e. >98%. Other than this the toxic response of test species was influenced mainly by the dose and exposure time. The acute toxic effect after ozonation was observed on V. fischeri and found that toxic effect of the secondary effluent was decreased after applied dose and exposure duration. Results supported the expectation that the process of Ozone might enhance the overall treatment efficiency of secondary effluent treatment.