Background Previous studies in the Bono Region (middle belt) of Ghana have reported persistent Onchocerca volvulus infection and associated morbidities after nearly three decades of ivermectin treatment. This study aimed to assess the usability, acceptability and cost of the Ov16 SD BIOLINE rapid diagnostic test (Ov16 RDT) in onchocerciasis surveillance activities in the middle belt of Ghana. Methodology A cross-sectional study was conducted in 6 endemic communities in the Tain District and Wenchi Municipality. A total of 254 individuals (54% females; median age (range)=31 (5–83) years), agreed to participate in Ov16 RDT (100%), skin-snip microscopy (37%) and nodule palpation (100%). Post-test interviews were conducted for all 94 participants tested by all three diagnostics. A cost analysis based on testing 400 people was performed. Principal findings Ov16 seroprevalence was 23.6% (60/254, 95%CI=18.8%–29.2%); microfilarial prevalence 11.7% (11/94, 95%CI = 6.7%–19.8%) and nodule prevalence 5.5% (14/254, 95%CI=3.3%–9.0%). The proportion of Ov16 seropositive females (43/136, 31.6%) was twice that of males (17/117, 14.5%). Among 5–9-year-olds, Ov16 seroprevalence was 11.1% (3/27), microfilarial prevalence 23.1% (3/13) and nodule prevalence 3.7% (1/27). For the 94 participants with all three tests, there was no association between the results of Ov16 RDT, skin-snip microscopy and/or nodule palpation. Most participants and technicians preferred Ov16 RDT because of being less painful and invasive, easier to use and faster. Had 400 participants been tested, the total cost per individual would be US$24 (Ov16 RDT) and US$74 (skin-snip microscopy). Conclusions Ov16 RDT is more acceptable and affordable (a third of the cost) compared to skin-snipping for surveillance activities in transmission hotspots in Ghana.