The ongoing development of new production methods may lead to the commercialization of -acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1-6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1β, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.