Aims: Acinetobacter baumannii is a global health problem, which threatens many healthcare settings. The current study aims to develop a detection assay for Ac. baumannii using unmodified gold nanoparticles (AuNPs). Methods and Results: Fifty-three Ac. baumannii clinical isolates were collected from Egyptian hospitals. Bacterial isolation and biochemical identification of isolates were carried out followed by DNA extraction using boiling method and PCR amplification of the 23S-16S rRNA intergenic spacer sequences (ITS). AuNPs were synthesized using citrate reduction method. Detection and optimization of Ac. baumannii amplicons using unmodified spherical AuNPs were performed using species-specific DNA oligonucleotide. The nano-gold assay was able to colorimetrically detect and distinguish Ac. baumannii from other Gram-negative bacteria. The turnaround time of the assay is about 2 h including sample treatment and amplification. The assay detection limit is 0.8125 ng of DNA. Conclusions: The developed colorimetric assay is sensitive, fast and reliable and can be used for identification of Ac. baumannii. Significance and Impact of the Study: There is a need to develop robust, rapid, and specific methods for detection of Ac. baumannii isolated from clinical specimens. The developed nanogold assay prototype allows sensitive, specific and rapid detection of amplified DNA of A. baumannii and represents a reliable diagnostic tool to aid routine laboratory identification of this pathogen.