Aim: Aim this study to the microbial and molecular analysis of environmental (air, water and surfaces) microbial contamination in Ukrainian dental clinics. Materials and Methods: This multicenter observational study was conducted between January 1, 2022, to December 31, 2024 in fifteen dental clinics located in five regions of Ukraine. Evaluation of the environmental contamination was performed according to microbial and molecular methods. Results: During study period in dental clinics tap water, dental unit water systems, and water from the bottles attached to the dental chair units (before clinical activity and during clinical activities), and surfaces of dental equipment, and air (during clinical activities) was heavily contaminated with potential pathogens of healthcare associated infections (HAIs). The bacteria identified were mainly of the Gram-positive and Gram-negative species. The main potentially HAI pathogenic of bacteria were Escherichia coli (13%), Pseudomonas aeruginosa (9.1%), Enterococcus faecalis (4%), Klebsiella pneumoniae (3.6%), Acinetobacter lwoffii (3.5%), Acinetobacter baumannii (3.3%), Klebsiella oxytoca (3.2%), Serratia marcescens (3.2%), Stenotrophomonas maltophilia (3.2%), Staphylococcus aureus (3.2%), Enterococcus faecium (3.1%), Enterobacter cloacae (3%), Enterobacter aerogenes (2.9%), Burkholderia cepacia (2.6%), Providencia stuartii (2.5%), Proteus mirabilis (2.2%), Proteus rettgeri (2.1%), and Streptococcus pneumoniae (2.1%). Conclusions: During study period in dental clinics tap water, dental unit water systems, and water from the bottles attached to the dental chair units (before clinical activity and during clinical activities), and surfaces of dental equipment, and air (during clinical activities) was heavily contaminated with potential pathogens of HAIs. Patients are exposed during dental practice to an infective risk.