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Theoretically, <i>Aspergillus</i> spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect underlines the lack of a good strategy for the cultivation and isolation of <i>Aspergillus</i> spp. In order to develop a complete analytical method to detect <i>Aspergillus</i> in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At <72 h, all test systems showed comparable sensitivity, about 1–2 conidia. However, the subculture analysis showed a suboptimal recovery for the methods, despite the positive growth and the visualization of the “<i>Aspergillus</i> balls” in the culture media. To investigate this issue, we studied three different subculture approaches: (i) the use of a sterile subculture unit, (ii) the use of a sterile subculture unit and the collection of a larger aliquot (100 µL), following vigorous agitation of the vials, and (iii) to decapsulate the bottle, withdrawing and centrifuging the sample, and aliquot the pellet onto SDA plates. Our results showed that only the third procedure recovered <i>Aspergillus</i> from all positive culture bottles. This work confirmed that our strategy is a valid and faster method to culture and isolate <i>Aspergillus</i> spp. from blood culture bottles.