Introduction: Oral hygiene is one's practice of keeping his teeth and mouth cavity clean in order of prevention of oral diseases. Toothbrush is the world's most common, most accepted and most used instrument for oral hygiene. Its everyday usage results in leftover microorganisms on the surface of the bristles. Research objectives: To identify and classify microorganisms from used toothbrushes and to define whether they are part of physiological oral microbiota or their origin is from another source. Methodes and materials: Toothbrushes were taken from persons of both sexes with a condition that they used them for 30 days or more. Heads of the toothbrushes were sawn off and dipped in liquid nutrient-rich media. Microorganisms from the media were placed on an universal and cromogenic agar plates and incubated for 24 to 48 hours. Further identification has been made in accordance with standard procedures used in microbiology. Results: 9 different bacterial species were identified from 14 toothbrushes: Klebsiella oxytoca, Enterobacter cloacae, Pantoea agglomerans, Pseudomonas aeruginosa, Escherichia coli, Serratia odorifera, Serratia marcescens, Enterococcus faecalis, Staphylococcus sppecies (coagulase negative). There was no significant statistical difference found (p > 0,05) in presence of Gram positive or Gram negative bacteria found on toothbrushes from smokers in relation to toothburshes from non-smokers (X2 = 1,938, df = 1, p = 0,1638). Also, no significant statistical difference (p > 0,05) in presence of bacterial species from other sources was found on toothbrushes covered with a safety cap in relation to those that weren't (X2 = 1,077, df = 1, p = 0,2994). There were no difference in other participant's habits or lifestyle. Conclusions: Domination of Gram negative bacteria that belong to human physiological gut microbiota indicates that contamination of toothbrushes from other sources has taken place. One of the possibilities is the transfer via bioaerosol generated by toilet systems. Further and more advanced research with a greater number of samples is needed in order to establish firm evidence of origin of the mentioned bacteria.