Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast  Schizosaccharomyces pombe  As opposed to many other alkylating agents, ENU only induces an S   N   1-type reaction with a low  s  constant ( s  = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.