The genital mycoplasmas are a unique group of inherently antibiotic-resistant sexually transmitted bacteria, often associated with non-gonococcal urethritis and bacterial vaginosis. The MYCO WELL D-ONE is a culture-based assay that aims to detect these organisms whilst concurrently screening them for antibiotic resistance. Urine and/or swabs from 856 informed and consented participants attending Welsh sexual health clinics were subjected to MYCO WELL D-ONE analysis, alongside qPCR and culture titration methodologies to determine sensitivity, specificity, PPV, NPV and accuracy. Resistance was confirmed by CLSI-compliant susceptibility testing and genetic mechanisms determined. The MYCO WELL D-ONE displayed a sensitivity and specificity of 91.98% and 96.44% for the detection of Ureaplasma spp., with sensitivity and specificity values of 78.23% and 98.84% for Mycoplasma hominis , compared with qPCR. Swabs harboured significantly greater bacterial loads than urine samples for both Ureaplasma spp. and M . hominis . Levofloxacin resistance rates, mediated by Ser83Leu mutation in ParC, for Ureaplasma spp. were 0.54%. Tetracycline resistance rates, mediated by tet (M), were 0.54% and 2% for Ureaplasma spp. and M . hominis , respectively; sequence analysis of tet (M)-positive Ureaplasma spp. and M . hominis strains isolated from a single individual confirmed separate resistance gene origins. The MYCO WELL D-ONE is a sensitive and specific assay for the detection of Ureaplasma spp. and M . hominis in genitourinary medicine samples, facilitating the accurate detection of these organisms within low-technology environments. While good for antibiotic resistance screening, accurate confirmation by MIC determination or molecular methods are required, and more optimally performed on urine samples.