The introduced eastern mosquitofish (Gambusia holbrooki) poses a great threat to the delicate ecosystem of Edgbaston Springs in central Queensland, home to the critically endangered, red-finned blue-eye (Scaturiginichthys vermeilipinnis). Here, competition for food and space is high, leading to direct competition among species, and as is often the case, the invaders are displacing natives. While earlier mosquitofish eradication programs have been successful, continued vigilance is necessary due to the potential for reinvasion after periods of intermittent inundation. This is difficult however, as early detection of new invasions (i.e. when initial density of invaders is small) can be problematic when relying on traditional monitoring methodologies. Furthermore, these methods are not desirable given the precarious nature of the habitat and its vulnerable endemics. As such, this study evaluated the efficacy of eDNA approach for noninvasively detecting mosquitofish at Edgbaston. Water samples from several springs, representing different times since eradication, were collected along with samples from pools where mosquitofish had never invaded. Extracted eDNA were subjected to PCR amplification and Illumina® Miseq sequencing, allowing us to potentially detect all species present in the sampled waterbodies. We were able to identify where mosquitofish DNA were present, however this included pools where they had been eradicated up to 6 months previously. We interpret this outcome as sensitivity of the sequencing method and that we were seeing residual eDNA persisting after mosquitofish removal. However, it was determined that with regular PCR, using Gambusia specific primers, we were able to detect current populations, while the method being not so sensitive as to amplify DNA > two months. However, casting a wide sequencing net using the Illumina platform has provided a data set that potentially contains representative DNA from all species that exist in this spring system – a very useful resource for continued conservation management at Edgbaston.