Objectives: The primary purpose of the recent experiment was to scrutinize the dissimilarity between single and multiple exposures by electrotherapeutic modalities to determine the development of Gram-positive and Gram negative bacteria spectrum. Material and methods: Bacterial strains employed in this study were Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonae and Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus saprophyticus and Streptococcus pyogenes. Fluence for Low level laser therapy (LLLT) (810 nm) was 40 J/cm2 for 80 s, for microwave (MWD) a dosage of 100-Watt with duration of 5 min and for magnetic field therapy (MT) duration of 30 min with 100% intensity was used. Results: Repeated Measures of analysis of variances (RANOVA) for within-subject effects was used to detect a global significant change within the means at dissimilar time points. The experiments of within-subjects revealed a significant difference within groups, df of (3, 40), F value of 39.38 and a p value less than 0.001, representing a significant variation between the three groups between pre and post exposures. There was a significant variation between single exposure and multiple exposures in the experimental sample’s pre-post between the four groups with df (1, 40) f value of 2943.69 and p value less than 0.001. Scanning and Transmission electron microscopy images were also taken into account to determine the extent of damage caused to the bacterial cells surface topography in Gram negative and Gram positive spectrums. Conclusion: The study demonstrated that single high exposure with the LLLT appears to have the most emphatic effect followed by exposure by MWD and MT.