Lenless microscopes that form images using simple, compact optics, along with associated computational algorithms provide several advantages: small, inexpensive form factors, large fields of view (FOV), and the ability to refocus an image after capture. To date, however, lensless microscopy has yet to be demonstrated for in vivo biological imaging. The primary challenge with in vivo bio-imaging is these dense, low-contrast scenes cannot be accurately reconstructed with existing lensless techniques. To overcome this challenge, we designed an optical phase mask that creates a point-spread function consisting of high-contrast contours with a broad spatial frequency spectrum. Here we show that this “contour” phase mask enables in vivo imaging where previous lensless imaging technologies have failed. As a proof of concept, we demonstrate 3D imaging of a behaving invertebrate, functional calcium imaging in mouse cortex, and in vivo imaging of microvasculature in the human oral mucosa. Together these results demonstrate the ability of lensless imaging to operate in realistic biological imaging applications ranging from pre-clinical animal models to clinical biomedical imaging. Overall, this “Bio-FlatScope” is a clinically relevant imaging technique with advantages of 3D computational refocusing in a low-cost, small form factor that is useful for low-resource settings and difficult-to-reach areas of the body.