Collagen is a body own protein that, due to its outstanding biocompatibility, is applicable as substrate for cell seeding in tissue engineering. Creating a substrate that mimics the characteristics of human tissue and, thus, allows for a sustainable proliferation of biological cells, requires a well-directed design of its mechanical properties. In the case of collagen, the exposure to ultraviolet (UV) light is a promising method for inducing stabilising or destabilising reactions that modify the mechanical properties, as for example, the tensile modulus. For enabling a targeted substrate design, the reaction of the collagen samples towards the UV light treatment needs to be understood and made predictable. Collagen fibrils, as small units of the collagen’s hierarchical structure, gained attention and are investigated regarding the response of their mechanical properties due to the exposure to UV light dependent on the kind of surrounding liquid. Atomic force microscopy (AFM) and Raman spectroscopy were applied to monitor the impact of UV light on collagen fibrils and hydrogels. Changes in the indentation modulus of single collagen fibrils were recorded for the exposure to UVA, UVB, or UVC light while being immersed in varied liquid environments in situ. Next, an experimental procedure for an AFM-based eased extraction of the tensile modulus from fibrillar structures as small as collagen fibrils was proposed, which was used for the acquisition of the changes in the tensile modulus of collagen fibrils, dependent on the combination of the applied kind of UV light and liquid environment. The observed modifications of the mechanical properties should be complemented by a description of structural changes induced by UV light irradiation for which Raman spectroscopy was used. Collagen fibrils responded to the exposure to UV light with a change in their mechanical properties and exhibited repeated patterns of behaviour dependent on the applied combination of UV light and the kind of liquid environment. A decrease in stability of up to 90% (tensile modulus) was detected for the immersion of the samples in deionised water. For gaining an increase in stability with advancing exposure time, the application of phosphate-buffered saline (PBS) was a necessary condition. A maximum averaged increase in indentation modulus of approximately 60% was seen during exposure of single collagen fibrils to UVB or UVC light. For the tensile tests, a fourfold increase of the tensile modulus was recorded as maximum value. The depicted dependence of the mechanical properties on the kind of liquid environment was confirmed by Raman spectroscopy on collagen hydrogels. The observed proliferation of biological cells on UV light exposed collagen hydrogels showed the applicability of the method.