Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second-harmonic generation (SHG) and third-harmonic generation images from ex vivo human skin and showed that a sub-40 fs, 1060-nm Yb-fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber-based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low-barrier-to-access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.