Electromagnetic waves are used in today lives and spreading the environmental factor. We need visible light to see, radio waves for telecommunication and x-ray for medical purpose. Electromagnetic radiations are used for its bactericidal effect. It affects the cellular mechanism, cell membrane and might increase or decrease the activity of protein. The species selected for study was Lactobacillus rahmnosus (MTCC 1423) and Lactobacillus plantarum (MTCC 9496). Lactobacillus is friendly type of bacteria that lives in our digestive system, urinary tract and used to treat many skin disorders as blisters, canker sores, acne. The culture were exposed to three different frequencies (7.50GHz, 7.62GHz, 6.41GHz) with four different time intervals 5mins, 10 min, 15min, 20min. For the growth test MRS agar used and serial dilution were made to 10-3 and 10-4. The result showed the significant decrease in the number of colonies (cfu/m) with increasing time and frequency, where as the antibiotic sensitivity test, antibiotics were Kanamycin (K30), Gentamicin (G50), Tetracycline (Te30), Norfloxacin (NX10) among all four antibiotics Norfloxacin (NX10) showed the maximum zone of inhibition and rest all have shown the random changes may be because of the activity of protein. The result of biochemical showed negative effect of electromagnetic radiation on Lactobacillus species.