Among the many inputs, nitrogen fertilizers are the main yield-limiting factor in agriculture. Liquid fractions of digestates can be a most promising substitute to synthetic nitrogen fertilizers, using little energy to turn waste into valuable fertilizers. In this study, the efficacy of five digestates from different origin (C, cow slurry; P, pig slurry; PCE, pig slurry, cow slurry, energy crops; SS, sewage sludge; W, organic fraction of municipal waste) were assessed as fertilizers for the cultivation of Lactuca sativa L., compared to traditional mineral fertilization. Digestates showed promising results as fertilizers for Lactuca sativa L., as yield and chemical parameters were overall comparable to the mineral fertilizer. Analysis of nitrogen evolution showed that most digestates showed higher nitrates in the substrates than the mineral fertilizers at earlier stages. Another topic investigated in the study is the effect of the digestates on the bacterial populations of the growth substrate, investigated through quantification and sequencing of 16S gene. These results varied based on the digestate considered, but, in general, an increase in biodiversity could be linked to use of digestates. These results suggest that digestates might become an alternative to mineral fertilizers, contributing to the circular economy and waste reduction.