An SYBR Green real-time polymerase chain reaction (PCR) assay was developed for Arcobacter detection in food and wastewater samples. The assay was applied to 36 chicken and 33 wastewater samples, and the results were compared with those obtained for conventional PCR, multiplex PCR, and culture isolation. Isolates were identified by multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment, and typed by randomly amplified polymorphic DNA. Arcobacter sp. was detected in 25 of the 26 chicken carcasses (96%) and in 4 of the 10 liver samples (40%) by real-time PCR. Twenty-five chicken samples were positive also by conventional PCR, but in most of them the detection was only possible after 48-h enrichment. Arcobacter butzleri was the most frequently detected species. Twenty-four Arcobacter isolates were obtained from chicken samples, where A. butzleri is the only identified species. All the wastewater samples (100%) were positive for Arcobacter sp. by real-time PCR without enrichment. A. butzleri and Arcobacter cryaerophilus were detected by multiplex PCR. Fifteen samples were found to be positive by culture. Thirty-six isolates were obtained; all of them were identified as A. butzleri by multiplex PCR. However, by PCR-restriction fragment length polymorphism, 34 were identified as A. butzleri, 1 as A. cryaerophilus, and another 1 as Arcobacter skirrowii. A great genetic heterogeneity was observed by randomly amplified polymorphic DNA-PCR profiling. The real-time PCR assay developed in this work showed better detection levels than conventional PCR, together with shorter times of testing samples. Therefore, it could be used as a rapid and accurate instrument for monitoring Arcobacter contamination levels in food and water samples.