Purpose To investigate the efficacy of adjunctive selective laser trabeculoplasty (SLT) in reducing 24-h intraocular pressure (IOP) fluctuations and nocturnal IOP peaks. Methods In this prospective interventional case series, 157 medically treated eyes of 157 patients with primary open-angle glaucoma (POAG) who were assigned SLT to further reduce IOP were consecutively included. Each patient had a complete glaucoma work-up and 24-h IOP monitoring (6 measurements, including one in the supine position) taken before and on average 6 months after SLT. The main outcome measures were the reduction of 24-h IOP fluctuations and nocturnal peak IOP. Secondary outcome measures were success rates, factors influencing the reduction of high 24-h IOP fluctuations and nocturnal peak IOP, complications, and severe adverse events. Results Medicated mean 24-h IOP (mmHg) was statistically significantly reduced from 15.1 ± 2.6 to 13.8 ± 2.4 (P < 0.001) and IOP fluctuations from 6.5 ± 2.7 to 5.4 ± 2.6 (P < 0.001) 6 months after SLT. Ninety-four eyes (59.9%) initially had high IOP fluctuations (more than 5 mmHg). These were reduced from 8.1 ± 2.3 to 5.6 ± 2.7 at 6 months (P < 0.001). Fifty-two eyes (55.3%) had fluctuations below 5 mmHg post-SLT which was defined as success. Fifty-one patients (32.5%) had nocturnal IOP peaks. In these cases, nocturnal IOP was reduced by 19.2% from 20.1 ± 3.4 to 16.2 ± 3.3 mmHg at 6 months (P = 0.001). Conclusions The current study demonstrates that adjunctive SLT not only reduces mean 24-h IOP in treated POAG patients, but also has an additional benefit in reducing IOP fluctuations and nocturnal peak IOP.