It has been advocated before that appearance prediction of unknown suspects from crime scene DNA, in the context of Forensic DNA Phenotyping (FDP), is mostly suitable for single source DNA samples, whereas FDP from DNA mixtures to which more than one person contributed, is viewed challenging. With this report on a murder case, we practically demonstrate the feasibility of appearance DNA prediction of an unknown suspect from a mixed crime scene trace, to which the unknown suspect and the known victim had contributed. From this two-person DNA mixture, we successfully predicted eye, hair and skin colour of the unknown suspect with the HIrisPlex-S system by applying targeted massively parallel sequencing (MPS). We argue that at least three factors benefit appearance DNA prediction of unknown suspects from mixed crime scene traces, which were met in this murder case: i) SNP genotype knowledge from reference DNA analysis for one of the two persons in the mixture (here the known victim), ii) about equal DNA contributions by both donors to the mixed crime scene stain, and iii) the use of MPS allowing quantitative SNP analysis. Moreover, we show that additionally analysing animal DNA in this mixed crime scene trace provides further investigative information. We envision that the investigative DNA strategy that we applied here for analysing a two-person mixed crime scene trace in a murder case, will be applied in the future to more criminal cases with two-person DNA mixtures, for instance sexual assault cases.