In this study, the microbial contamination of smartphones from Italian University students was analyzed. A total of 100 smartphones classified as low, medium, and high emission were examined. Bacteria were isolated on elective and selective media and identified by biochemical tests. The mean values of cfu/cm were 0.79 ± 0.01; in particular, a mean of 1.21 ± 0.12, 0.77 ± 0.1 and 0.40 ± 0.10 cfu/cm was present on smartphones at low, medium, and high emission, respectively. The vast majority of identified microorganisms came from human skin, mainly Staphylococci, together with Gram-negative and positive bacilli and yeasts. Moreover, the main isolated species and their mixture were exposed for 3 h to turned on and off smartphones to evaluate the effect of the electromagnetic wave emission on the bacterial cultivability, viability, morphology, and genotypic profile in respect to the unexposed broth cultures. A reduction rate of bacterial growth of 79 and 46% was observed in Staphylococcus aureus and Staphylococcus epidermidis broth cultures, respectively, in the presence of turned on smartphone. No differences in viability were observed in all detected conditions. Small colony variants and some differences in DNA fingerprinting were detected on bacteria when the smartphones were turned on in respect to the other conditions. The colonization of smartphones was limited to human skin microorganisms that can acquire phenotype and genotypic modifications when exposed to microwave emissions.