Legionella pneumophila virulence is controlled in a growth phase-dependent manner by a complex regulatory network involving several two-component systems, small regulatory RNAs and the translational CsrA regulator. Here, we address the additional role of Nucleoid-associated proteins (NAP) regulators in this network, by investigating the regulatory functions of the three Fis paralogs (Fis1, Fis2, Fis3), a unique feature among bacteria, in the infection cycle of L. pneumophila . Specifically, we show that deletion of fis1 has a major impact on L. pneumophila virulence, and that deletion of fis2 enhances the intensity of this phenotype. Consistently, RNA-seq analysis and reporter gene fusions demonstrate the predominant role of Fis1 in the regulation of many virulence-related genes, including those involved in the flagellum, pili biosynthesis, and Dot/Icm type 4 secretion machinery, as well as several genes encoding Dot/Icm effectors. Both Fis1 and Fis2 bind to AT-rich motifs upstream their target genes, but Fis1 with higher affinity than Fis2. Importantly, Fis1 and Fis2 would be capable of forming heterodimers that could bind with variable affinity to this AT-rich motif. It is also important to note that the three Fis proteins are not produced at the same time and in the same amounts. We therefore hypothesize that the duplication of fis genes in L. pneumophila is not simply a back-up system to compensate for potentially deleterious mutations in a fis gene, but rather a means to fine-tune the expression of targeted genes, particularly virulence genes. IMPORTANCE Appropriate control of virulence gene expression is crucial to the success of bacterial infection. Nucleoid-associated protein regulators, including Fis proteins, have been shown to participate in the virulence of several human pathogens. The importance of our discovery lies in the fact that L. pneumophila possesses three non-homologous Fis proteins instead of just one. We demonstrate that Fis1 and Fis2 are not functional duplicates of each other. On the contrary, Fis1 and Fis2 are synthesized neither simultaneously nor in equal amounts during the bacterial growth phase, and they cooperate to regulate virulence gene expression by targeting similar AT-rich motifs, albeit with distinct affinity, and by being capable of forming heterodimers. Taken together, our data suggest that the high conservation of fis gene duplication results from the need for fine-tuned control of Legionella virulence in response to its different environmental and human hosts, rather than from functional redundancy to circumvent deleterious fis mutations.