AbstractAs a class, seminal fluid proteins are expected to exert strong effects on mating partners due to the selection pressures of sperm competition and sexual conflict. But because of the complexity of this secretion, linking specific proteins to downstream effects on own fitness—via manipulating the reproductive behavior, physiology, and ultimately the sperm utilization of mating partners—is not straightforward. Here, we adopted a systematic gene knockdown approach to screen for seminal fluid‐mediated fitness effects in the simultaneously hermaphroditic flatwormMacrostomum lignano. We focused on 18 transcripts inM. lignanoseminal fluid, testing how their RNA interference‐induced knockdown impacted on three aspects of donor (male) reproductive success: (a) fertility (offspring production of the partner); (b) defensive sperm competitive ability,P1; and (c) offensive sperm competitive ability,P2. In general, the knockdown of most individual transcripts appeared to have only a minor impact on male reproductive success, though we found evidence that the knockdown of up to five different transcripts impacted on fertility; the knockdown of two other transcripts resulted in reducedP2; and knockdown of a further transcript actually increasedP2. We thus identify a number of candidate seminal fluid transcripts that appear to modulate offspring production and sperm competitiveness inM. lignano. That only a minority of transcripts exhibit such a pattern likely reflects both the difficulty of accurately estimating sperm competitiveness and the functional redundancy of seminal fluid.