A new protocol for the treatment of Onychomycosis with laser irradiation was developed and used on 192 subjects with multiple nails affected by Onychomycosis, as confirmed by PAS, KOH and culture tests. A novel Nd:YAG 1064nm laser with a 650 microsecond pulse duration and a peak power of 318 joules/cm2 or 15,385 watts per pulse was used at two clinical sites over a period of 18 months. The protocol included two laser treatment sessions spaced approximately 4 weeks apart. Then, at a point 3 months after the second laser treatment session, each subject was again administered the PAS, KOH and culture tests, and 178 or 92.7% tested negative for the fungus. The two-treatment laser protocol was repeated on the 14 remaining subjects and 11 of them were clear of all fungal infection after two additional laser treatments. Therefore, 98.4% of all subjects have shown negative culture 3 months after completion of their laser treatment course. Toes were pre-cooled with ice and all subjects reported the laser treatment as either painless or highly tolerable with an occasional sensation of heat. There were no side effects of treatment, and all subjects were satisfied with the results. This clinical study demonstrates that Onychomycosis can be effectively and safely treated with a 650 microsecond Pulsed Nd: YAG 1064nm Laser.