Healthcare-associated infections (HAIs) are infections related to receiving medical care. HAIs are responsible for an excess of morbidity and mortality among hospitalized patients. Though most HAIs rates are on the decline, Clostridium difficile infection rates are at an all-time high, primarily due to the persistence of C. difficile spores in the environment. In the United States, Clostridium difficile-related mortality rates per million have increased from 5.7 in 1999 to 23.7 in 2004, with an estimated 26,642 deaths due to Clostridium difficile infections (CDIs). Clostridium difficile is transmitted via the fecal-oral route or aerosolized endospores, but it can also be transmitted from high touch surfaces in healthcare facilities, such as door handles, bed rails, and bed pans contaminated with C. difficile spores. Various methods of detection have been established since the 1970s, but they have limitations, such as cost, time, and availability. The use of a molecular method of detection, such as polymerase chain reaction (PCR), could provide more rapid and sensitive results for the detection of Clostridium difficile. The objective of this study was to determine the presence of Clostridium difficile pre- and post-decontamination of patients’ rooms in a healthcare facility environment using culture and PCR analysis of surface samples. No culturable C. difficile were detected; however, the culture analysis results showed a significant difference between the number of facultative and anaerobic bacteria in pre-decontamination samples and post-decontamination samples (Z = -5.852, p = 0.000). Of the 128 samples tested using PCR analysis, five samples were positive for Clostridium difficile DNA (3.9%); three were from pre-decontamination samples and two were from post-decontamination samples. Reducing the rate of transmission of Clostridium difficile infections in hospitals is dependent on a number of factors (e.g., proper use of antibiotics, environmental decontamination, and proper hand-hygiene). The results of this study indicate decontamination methods used at these facilities were effective in preventing environmental contamination of hospital rooms with facultative and anaerobic bacteria such as, C. difficile