Purpose: This study aimed to assess the persistent presence of microorganisms on patient-derived dental impressions and gypsum casts, while highlighting important human pathogens such as Candida, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa. Materials and Methods: The practices and opinions regarding cross-infection control from 59 general dentists in Japan were obtained via a questionnaire. Alginate impressions were made from 56 patients. Using a brain heart infusion agar medium, impression and imprint cultures were carried out to visualize the microbial contamination on the surfaces of the impressions and gypsum casts, respectively. The colonies on the surfaces of the 30 impression cultures and 26 imprint cultures were collected by swabbing and then inoculated onto selective agar plates to detect streptococci, staphylococci, Candida, MRSA, and P aeruginosa. Results: The questionnaire showed that only 54% of general dentists had a cross-infection policy in their dental clinics, and only 30% to 40% were aware of the possible persistence of MRSA or P aeruginosa on impressions and gypsum casts. The impression/imprint cultures grew a large number of visible bacterial colonies on all of the impression/gypsum cast samples investigated. Selective agar cultures demonstrated the presence of streptococci (100, 100%), staphylococci (56.7, 65.4%), Candida (30, 46.2%), MRSA (26.7, 15.4%), and P aeruginosa (6.7, 7.7%) on the impressions and the gypsum casts, respectively. Conclusions: This investigation showed that patient-derived dental impressions and gypsum casts are contaminated with numerous microbes, including Candida, MRSA, and P aeruginosa, which are known pathogens responsible for nosocomial and/or life-threatening infection in the immunocompromised host.