Background Smartphone-based fundus imaging allows for mobile and inexpensive fundus examination with the potential to revolutionize eye care, particularly in lower-resource settings. However, most smartphone-based fundus imaging adapters convey image quality not comparable to conventional fundus imaging. Objective The purpose of this study was to evaluate a novel smartphone-based fundus imaging device for documentation of a variety of retinal/vitreous pathologies in a patient sample with wide refraction and age ranges. Methods Participants’ eyes were dilated and imaged with the iC2 funduscope (HEINE Optotechnik) using an Apple iPhone 6 in single-image acquisition (image resolution of 2448 × 3264 pixels) or video mode (1248 × 1664 pixels) and a subgroup of participants was also examined by conventional fundus imaging (Zeiss VISUCAM 500). Smartphone-based image quality was compared to conventional fundus imaging in terms of sharpness (focus), reflex artifacts, contrast, and illumination on semiquantitative scales. Results A total of 47 eyes from 32 participants (age: mean 62.3, SD 19.8 years; range 7-93; spherical equivalent: mean –0.78, SD 3.21 D; range: –7.88 to +7.0 D) were included in the study. Mean (SD) visual acuity (logMAR) was 0.48 (0.66; range 0-2.3); 30% (14/47) of the eyes were pseudophakic. Image quality was sufficient in all eyes irrespective of refraction. Images acquired with conventional fundus imaging were sharper and had less reflex artifacts, and there was no significant difference in contrast and illumination (P<.001, P=.03, and P=.10, respectively). When comparing image quality at the posterior pole, the mid periphery, and the far periphery, glare increased as images were acquired from a more peripheral part of the retina. Reflex artifacts were more frequent in pseudophakic eyes. Image acquisition was also possible in children. Documentation of deep optic nerve cups in video mode conveyed a mock 3D impression. Conclusions Image quality of conventional fundus imaging was superior to that of smartphone-based fundus imaging, although this novel smartphone-based fundus imaging device achieved image quality high enough to document various fundus pathologies including only subtle findings. High-quality smartphone-based fundus imaging might represent a mobile alternative for fundus documentation in clinical practice.